The characterization of the T cell-inductive hormone, thymopoietin, will be further advanced through functional analysis of a biochemically and antigenically related (in the radioimmunoassay cross-reacting) but functionally distinct product found in murine spleen and lymphonode (but not in other tissues). This product, named splenin, differs from thymopoietin only at position 34, Asp for bovine thymopoietin and Glu for bovine splenin. Synthetic peptides corresponding to residues 32-36, called thymopentin (TP-5) and splenopentin (SP-5) reproduce biological activities of thymopoietin and splenin, respectively. Thus, thymopoietin and TP-5 induce the phenotypic differentiation of T precursor cells in vitro while inhibiting differentiation of B precursor cells. In contrast, splenin and SP-5 induce both T and B precursor cells. This new molecule and TP-5 analogs modified with respect to their sensitivity towards protein activity in vivo and in vitro are characterized for their functional activity in vivo.